Reactivity, electrochemical, and spectroscopic studies of type 2 copper-depleted Rhus vernicifera laccase

Wynn, R. Max; Knaff, David B.; Holwerda, Robert A.

doi:10.1021/bi00297a011

Cited by 11 publications

(1 citation statement)

References 36 publications

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“…The band at 628 nm, due to the copper ion of the protein active site, did not change, demonstrating that the T1 copper site was unaffected by metal binding to the Cterminal 6xHis-tag (data not shown). Notably, following addition of Cu 2+ ions, the spectrum showed an absorption increase in the 300-350 nm region, which has been previously observed for hexapeptide/Cu 2+ complexes [15,26] and for various blue oxidases containing a type 3 copper center [27][28][29]. In all cases, the copper ion was coordinated by the imidazole rings of two or three histidines while the remaining coordinating positions were free or occupied by solvent molecules.…”

Section: Metal-ion Binding To the 6xhis-tagged Azurins 331 Uv-visibsupporting

confidence: 63%

Excitation-Energy Transfer Paths from Tryptophans to Coordinated Copper Ions in Engineered Azurins: a Source of Observables for Monitoring Protein Structural Changes

Rocco

Bernini

Borsari

et al. 2016

Zeitschrift Für Physikalische Chemie

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The intrinsic fluorescence of recombinant proteins offers a powerful tool to detect and characterize structural changes induced by chemical or biological stimuli. We show that metal-ion binding to a hexahistidine tail can significantly broaden the range of such structurally sensitive fluorescence observables. Bipositive metal-ions as Cu2+, Ni2+ and Zn2+ bind 6xHis-tag azurin and its 6xHis-tagged R129W and W48A-R129W mutants with good efficiency and, thereby, quench their intrinsic fluorescence. Due to a much more favourable spectral overlap, the 6xHis-tag/Cu2+ complex(es) are the most efficient quenchers of both W48 and W129 emissions. Based on simple Förster-type dependence of energy-transfer efficiency on donor/acceptor distance, we can trace several excitation-energy transfer paths across the protein structure. Unexpected lifetime components in the azurin 6xHis-tag/Cu2+ complex emission decays reveal underneath complexity in the conformational landscape of these systems. The new tryptophan emission quenching paths provide additional signals for detecting and identifying protein structural changes

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Section: Metal-ion Binding To the 6xhis-tagged Azurins 331 Uv-visibsupporting

confidence: 63%

Excitation-Energy Transfer Paths from Tryptophans to Coordinated Copper Ions in Engineered Azurins: a Source of Observables for Monitoring Protein Structural Changes

Rocco

Bernini

Borsari

et al. 2016

Zeitschrift Für Physikalische Chemie

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Substitution of the Axial Type 1 Cu Ligand Affords Binding of a Water Molecule in Axial Position Affecting Kinetics, Spectral, and Structural Properties of the Small Laccase Ssl1

Olbrich,

Mielenbrink,

Willers

et al. 2024

Chemistry A European J

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Multicopper oxidases use Cu ions as cofactors to oxidize various substrates. High reduction potential at Type 1 Cu is considered as crucial for effective catalysis. Previous studies have shown that replacing the axial methionine ligand of the Type 1 Cu with leucine or phenylalanine leads to an increased reduction potential, but not always to higher enzyme activity. Here we present a study of six variants of the small laccase Ssl1 from Streptomyces sviceus, where the axial methionine ligand was substituted and the effect of the axial ligand on reduction potential, activity, spectral properties and structure was investigated. Absorption, electronic circular dichroism and EPR spectra revealed the presence of a stronger coordinating axial ligand like oxygen, which influences the electronic and catalytic properties more than the nature of the amino acid side chain. The crystal structures of the Ssl1 variants were solved, which show that none of the amino acids side chains coordinates to the Cu. Instead, a water molecule is found in the axial coordination site, which support the spectroscopic data. Our findings highlight the importance of combining structural and spectroscopic methods to investigate the effect of amino acid exchange on multicopper oxidases.

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Reduction thermodynamics of the T1 Cu site in plant and fungal laccases

et al. 2005

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The thermodynamic parameters for reduction of the type-1 (T1) copper site in Rhus vernicifera and Trametes versicolor laccases and for the derivative of the former protein from which the type-2 copper has been selectively removed (T2D) have been determined with UV-vis spectroelectrochemistry. In all cases, the enthalpic term turns out to be the main determinant of the Eo' of the T1 site. Also the difference between the reduction potentials of the two laccases is enthalpy-based and reflects differences in the coordination features of the T1 sites and their protein environment. The T1 sites in native R. vernicifera laccase and its T2D derivative show the same Eo', as a result of compensatory differences in the reduction thermodynamics. This suggests that removal of the type-2 (T2) copper results in modification of the reduction-induced solvent reorganization effects, with no influence in the structure of the multicopper protein site. This conclusion is supported by NMR data recorded on the native, the T2D, and Hg-substituted T1 derivatives of R. vernicifera laccase, which show that the T1 and T2/T3 sites are largely noninteracting.

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Reactivity, electrochemical, and spectroscopic studies of type 2 copper-depleted Rhus vernicifera laccase

Cited by 11 publications

References 36 publications

Excitation-Energy Transfer Paths from Tryptophans to Coordinated Copper Ions in Engineered Azurins: a Source of Observables for Monitoring Protein Structural Changes

Excitation-Energy Transfer Paths from Tryptophans to Coordinated Copper Ions in Engineered Azurins: a Source of Observables for Monitoring Protein Structural Changes

Substitution of the Axial Type 1 Cu Ligand Affords Binding of a Water Molecule in Axial Position Affecting Kinetics, Spectral, and Structural Properties of the Small Laccase Ssl1

Reduction thermodynamics of the T1 Cu site in plant and fungal laccases

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